Pharmaceutical composition comprising bee venom-phospholipase A2 (BV-PLA2) for treating or preventing diseases related to degradation of abnormal regulatory T cell activity

ABSTRACT

The present invention relates to a pharmaceutical composition for treating or preventing a disease related to abnormal suppression of regulatory T cell activity comprising a polypeptide comprising a bee venom-PLA2 amino acid sequence exclusive of a leader sequence as an active ingredient. The secretory bee venom-phospholipase A2 of the present invention activates a regulatory T cell and suppress a differentiation of Th1/Th7. Therefore, the present polypeptide can be used as a pharmaceutical composition for treating or preventing a disease related to abnormal suppression of regulatory T cell activity, i.e. autoimmune diseases, allergic diseases, or neurodegenerative diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/381,095, filed Dec. 8, 2014, which is a U.S. National StageApplication filed under 35 U.S.C. 371 and claims priority toInternational Application No. PCT/KR2012/004394, filed Jun. 4, 2012,which application claims priority to Korean Application No.10-2012-0019925, filed Feb. 27, 2012, the disclosures of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition fortreating or preventing diseases related to abnormal suppression ofregulatory T cell activity containing bee venom-phospholipase A2(BV-PLA2) as an active ingredient.

BACKGROUND ART

One of the most important features in all normal subjects is that theydo not harmfully react against antigenic materials forming a self whilethey are capable of recognizing, reacting to, and removing numerousnon-self antigens. The above biological phenomenon of not beingresponsive to self antigens is called immunologic unresponsiveness ortolerance. Self-tolerance occurs when the lymphocytes which may havespecific receptors for self antigens are removed or when the reactivefunction that responds to self antigens after being contacted isinactivated. When there is a problem in inducing or maintainingself-tolerance an immune response occurs against self antigens, and thediseases caused thereby is called an autoimmune disease.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease inthe central nervous system affecting more than a million peopleworldwide. MS causes substantial disorders due to deficiency insensation and locomotion, autonomy and neurocognitive functions. Themechanism of developing the disease generally appears to be anautoimmune pathology in which CD4⁺ T helper 1 cells (Th1: IFN-γproducing CD4⁺ T cells), T helper 17 cells (Th17: IL-17A producing CD4⁺T cells) and regulatory T cells (Treg) play important roles. Manyclinical and pathological characteristics of the experimental autoimmuneencephalomyelitis (EAE), which is an animal model of human MS, showproximal similarity to that of MS. Accordingly, EAE has been generallyused as an experimental model system for studying the mechanism of MSdevelopment and for testing the efficacy of potential therapeutic agentsfor MS. Therapeutic agents for controlling diseases such as IFN-β andglatiramer acetate have been widely used for MS treatment, showingadvantageous effects. However, due to limitations on therapeutic methodsmany MS patients have been looking for selective alternative therapies.Allegedly, about 50% to 75% of MS patients are using at least onecomplementary and alternative medicinal therapy.

An allergic disease refers to a disease caused by a disorder in immunesystem in which a substance non-harmful to normal people causeshypersensitivities with various symptoms to particular people. Asubstance that causes an allergic disease is called an allergen orantigen, and pollens, antibiotics, drugs, dusts, foods, cold air,sunlight, etc., may causes the allergies. The symptoms of the allergicdiseases include hives, sneezing, pruritus, rhinorrhea, coughs, hayfever, ocular hyperemia, eczema, rashes, etc. Representative allergicdiseases are allergic asthma, which accompany symptoms such as airwayconstriction, increase in the secretion of mucus liquid in the lungs,dyspnea, and coughs, and additionally may include atopic dermatitis,conjunctivitis, rhinitis, and ulcerative colitis.

Neurodegenerative diseases are associated with symptoms such asdegeneration, loss of functions, and often apoptosis of neurons. Sincethese symptoms are progressive they are often highly destructive unlikethe neurodegenerative diseases. Patients with neurodegenerative diseasesmay experience extreme deterioration in their cognitive or motorperformance. Accordingly, the quality of their lives and expectationthereto may be considerably deteriorated.

Parkinson's disease (PD) is a representative progressiveneurodegenerative disease characterized by loss of dopaminergic (DA)neurons in substantia nigra (SN), and shaking, rigidity, slowness ofmovement, and bradykinesia due to decrease in dopamine in striatum(STR). PD is a sporadic disease whose pathogenesis has not beenidentified. According to the evidence accumulated so far,neuroinflammation appears to play an important role in the pathogenesisof PD. The primary trigger of neuroinflammation is activated microglia,which are innate immune cells of the central nervous system (CNS)discovered in degenerative DA neurons and therearound. Microglia aredramatically activated by responding to neuronal damage, and ROS and/orproduce various potential neurotoxins including proinflammatorycytokines. Until recently, the role of the adoptive immune system hasbeen increasingly emphasized in PD pathogenesis. Examples of therapeuticdrugs for treating PD include L-dopa preparation, dopamine agonists,anticholinergics, Eldepryl (depreyl), etc. Most of these drugs areinvolved in regulating the symptoms of PD rather than treating itscause, and thus they should be administered continuously withoutcessation. However, long-term administration of these drugs may lead todrug intoxication. For example, anticholinergics may cause disorders inautonomic nervous system or abnormalities in mental functions and thusits long-term administration to people of senile age are limited.Additionally, the efficacies of L-dopa preparations may progressivelydeteriorate as the duration of its administration becomes long, and mayalso incur adverse effects such as twisting of the body and involuntarymovements of hands or legs. Accordingly, in order to prevent the adverseeffects, active efforts have been made to develop a therapeutic agentderived from natural substances for PD treatment. For example,pharmaceutical compositions containing a Scutellariae Radix extract (KRPatent Application Publication No. 2001-0081188), a Beauveria Bassiana101A extract (KR Patent Application Publication No. 2004-0012396), apeach leaf extract (KR Patent Application Publication No. 2010-0060949),etc., as active ingredients have been disclosed. Although the componentsderived from natural substances have no adverse effects they aredisadvantageous due to low therapeutic effects for PD treatment.

Since the introduction of the regulatory T cell concept on early 1970sby Gershon based on the possibility of the presence of T cells capableof controlling and inhibiting the effector functions of palliative Tcells (conventional T cells), and its first disclosure (R. K Gershon andK Kondo, Immunology, 1970, 18: 723-37), studies have been focused on theelucidation of biological characteristics and functions of regulatory Tcells in many fields of immunology.

In particular, since Sakaguchi suggested in 1995 that CD25 can act as animportant phenotypic marker for naturally-occurring CD4⁺ regulatory Tcells (S. Sakaguchi et al., J. Immunol., 1995, 155: 1151-1164), thestudies have focused on the roles and importance of regulatory T cellsin inducing peripheral tolerance regarding self antigens.

Bee venom is an alternative medicine widely used for the treatment of afew immune diseases, in particular rheumatoid arthritis. The existingstudies have disclosed that bee venom treatment can alleviate rheumatoidarthritis and has an anti-inflammatory effect in humans and experimentalanimals. Additionally, although there are evidence-based descriptivereports on the alleged improvement of neuropathy symptoms by MS patientswho have received multiple repeated bee venom acupunctures a conclusivedecision on the bee venom efficacy has not been made due to lack ofdetailed studies, and currently there is almost no evidence supportingthe use of bee venom in MS treatment.

Meanwhile, although bee venom is widely used as an alternative medicine,considering the risk of hypersensitivity, shock response, etc., in usingbee venom, it is necessary to identify specific active ingredients andthe working mechanism of bee venom, and to use it as a more purifieddrug.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

Accordingly, the present inventors have endeavored to study the activeingredients of bee venom having therapeutic effects on diseases andtheir working mechanisms, and found that phospholipase A2 (PLA2) amongthe various bee venom ingredients, in particular a polypeptidecontaining a secretory bee venom-PLA2 amino acid sequence exclusive of aleader sequence, can activate regulatory T cells thereby exhibitingeffects of preventing and treating autoimmune diseases, allergicdiseases, or neurodegenerative diseases.

Technical Solution

An objective of the present invention is to provide a pharmaceuticalcomposition for treating or preventing diseases related to abnormalsuppression of regulatory T cell activity containing a polypeptide whichincludes a bee venom-PLA2 (BV-PLA2) amino acid sequence exclusive of aleader sequence.

Advantageous Effects

According to the present invention, a polypeptide including a beevenom-PLA2 amino acid sequence exclusive of a leader sequence caninhibit Th1/Th17 differentiation while activating regulatory T cells.Accordingly, the polypeptide of the present invention can be useful as apharmaceutical composition for preventing or treating diseases relatedto abnormal suppression of regulatory T cell activity, i.e., autoimmunediseases, allergic diseases, and neurodegenerative diseases, without therisks of using unpurified and unisolated bee venom.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram briefly illustrating an experimentprotocol for an allergic asthma animal model including the onset timewhen the experimental mice were sensitized with OVA and the time whenthey were treated with BV-PLA2 (secretory type).

FIG. 2A-E shows graphs illustrating the effect of bee venom (BV) on theexpression of Foxp3 of CD4⁺CD25⁺Foxp3⁺ Treg and CD4⁺CD25⁺ Treg. (A) and(B) show the cytometry analysis results of the splenocytes ofFoxp3^(EGFP) C57BL/6 mice after treating them with variousconcentrations of bee venom (0, 0.001, 0.01, 0.1, 1, and 10 μg/ml) for24 hours followed by staining with CD4 and anti-CD25 antibodies. (A)shows the analysis results of CD25 and Foxp3 positive cells after gatingon CD4⁺ T cells, and (A) shows the analysis results of expression rateof Foxp3^(EGFP) after gating on CD4⁺CD25⁺ T cells. (C) through (E) showthe results of flow cytometry analysis of murine cells isolated from aFoxp3^(EGFP) C57BL/6 mouse after treating them with bee venom (1 μg/ml)for 24 hours followed by staining with anti-CD4 and anti-CD25antibodies. (C) shows the result of flow cytometry analysis of CD4⁺ Tcells, (D) shows that of the CD4⁺CD25⁻ T cells, and (E) shows that ofthe CD4⁺CD25⁺ Treg cells. The numbers within the dot plots of (A), (C),and (D) respectively represent the percentage of cells belonging to thecorresponding quadrant. The percentages of (B) and (E) represent aproportion of the gated Foxp3^(high) cells in the CD4⁺CD25⁺ Treg.Experiments were repeated 3 times and one representative graph is shown,wherein data is shown via mean±standard error of the mean (SEM)(*p<0.05, ***p<0.001 vs. a PBS-treated cell group).

FIG. 3A-D shows graphs illustrating the effect of bee venom (BV) on theproportion of various in vivo cell populations. Foxp3^(EGP) C57BL/6 miceor C57BL/6 mice were administered with bee venom (0.01, 0.1, 1, and 10mg/kg body weight) or an equal volume of brine daily for 5 days viaintraperitoneal injection. The single-cell suspension of splenocytes wasstained with fluorescence-labeled CD4, CD8a, CD19 and CD25 antibodies.(A) shows graphs illustrating the percentages of CD4⁺CD25⁺ Treg measuredin murine splenocytes. (B) shows graphs illustrating the expressionrates of Foxp measured after gating on the CD4⁺CD25⁺ Treg. Thepercentage is a proportion of Foxp3^(high) cells being gated on theCD4⁺CD25⁺ Treg. (C) shows the expression rates of CD25 and/or Foxp3measured after gating on the CD4⁺ T cells. The numbers within the dotplots of (A) and (C) respectively represent the percentage of cellsbelonging to the corresponding quadrant. (D) represents the percentageof the CD4⁺CD25⁺Foxp3⁺ Treg in the CD4⁺ T cells, or the percentage ofthe CD4⁺ T cells in the splenocytes, CD8⁺ T cells, and B cells. The datais indicated via mean±SEM ((ns; not significant), *p<0.05, **p<0.01,***p<0.001 vs. a brine-treated group).

FIG. 4 shows a graph illustrating the in vitro inhibitory effect of beevenom against antigen-specific growth. The splenocytes of anexperimental autoimmune encephalomyelitis (EAE)-induced C57BL/6 micewere stimulated with MOG₃₅₋₅₅ peptide and counted 72 hours thereafter.The data is indicated via mean±SEM ((ns; not significant), #p<0.001 vs.non-stimulated cells, ***p<0.001 vs. PBS-treated cells).

FIG. 5A-D shows graphs illustrating the effects of bee venom onalleviating EAE via inhibition of Th1/Th17 differentiation. The C57BL/6mice were immunized with MOG₃₅₋₅₅ peptide. (A) shows a graphillustrating mean clinical disease scores measured by monitoring themouse for 35 days after treating the mouse with bee venom (1 mg/kg bodyweight) from day 0 to 9th day after immunization (n=5/group). In (B)through (D), the mice were treated with bee venom (1 mg/kg body weight)from the 14th day to the 23^(rd) day after immunization (n=12/group).(B) shows a graph illustrating mean clinical disease scores measured bymonitoring the mouse for 35 days (n=12/group), (C) shows Th1/Th17percentages measured after re-stimulating the splenocytes with PMA (50ng/ml) and ionomycin (1 μg/ml) for 5 hours in the presence of monensin(n=11/group), and (D) shows cytokine concentration in the serum measuredafter collecting on the 24^(th) day (n=8/group). shows Th1/Th17percentages measured after re-stimulating the splenocytes with PMA (50ng/ml) and ionomycin (1 μg/ml) for 5 hours in the presence of monensin(n=11/group), and (D) shows cytokine concentration in the serum measuredafter collecting on the 24^(th) day (n=8/group). The data is indicatedvia mean±SEM (*p<0.05, **p<0.01 vs. brine-treated group).

FIG. 6A-C shows the effect of bee venom on alleviating EAE blocked dueto the CD4⁺CD25⁺ Treg cell depletion. C57BL/6 mice were immunized withMOG₃₅₋₅₅ peptide. Anti-CD25 antibodies were administered on the 13^(th)day and the 19^(th) day (respectively, the early stage treated with beevenom and an intermediate stage) of Treg depletion via intraperitonealinjection (A) shows a graph illustrating mean clinical disease scoresmeasured by monitoring for 24 days. (B) and (C) show the results ofanalysis after sacrificing the mice on the 24^(th) day afterimmunization (B) shows Th1/Th17 percentages measured afterre-stimulating the splenocytes with PMA (50 ng/ml) and ionomycin (1μg/ml) for 5 hours in the presence of monensin (n=8/group), and (C)shows cytokine concentration in the serum measured after collecting onthe 24^(th) day (n=5/group). The data is indicated via mean±SEM (ns; notsignificant).

FIG. 7A-C shows the effect of bee venom on the increase of CD4⁺Foxp3⁺Treg and the decrease of Th1/Th17. Foxp3⁺ Treg or Th1/Th17 was analyzedby gating on human CD4⁺ T cells. (A) shows the proportional ratio ofCD4⁺Foxp3⁺ cells, which were treated with bee venom (1 μg/ml) for 24hours after separating PBMC and gating, indicated in percentages. (B-C)was treated with bee venom (1 μg/ml) and anti-CD3/28 antibodies for 24hours after separating PBMC. Then, the cells were re-stimulated with PMA(50 ng/ml) and ionomycin (1 μg/ml) for 5 hours in the presence ofmonensin, and the result is shown in terms of population distribution.The numbers within the dot plots represent the percentage of cellsbelonging to the corresponding quadrant.

FIG. 8A-B shows the comparative results of various bee venom componentson immune-regulation inducing effect. The splenocytes of theFoxp3^(EGFP) C57BL/6 mice were treated with bee venom or itscomponent(s) for 24 hours, stained with anti-CD4 and anti-CD25antibodies, and then analyzed via flow cytometry (A). The CD4⁺ T cellswere gated, and CD25 and Foxp3 positive cells were analyzed. The numberswithin the dot plots represent the percentage of cells belonging to thecorresponding quadrant (B). The data is indicated via mean±SEM (*p<0.05,**p<0.01 vs. a PBS-treated group).

FIG. 9A-B shows the results of immune-regulation induced by humansecretory BV-PLA2 (bee venom-phospholipase A2; secretory beevenom-PLA2). Human PBMC was re-stimulated with anti-CD3/28 antibodiesand bee venom or BV-PLA2 for 24 hours. Then, the cells werere-stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 5 hours inthe presence of monensin. the CD4⁺ T cells were gated, and Th1/Th17 wasanalyzed (A). The numbers within the to dot plots represent thepercentage of cells belonging to the corresponding quadrant (B).

FIG. 10A-B shows in vitro CD4⁺CD25⁺Foxp3⁺ Treg population ofsplenocytes. The splenocytes stimulated with anti-mouse CD3 antibodiesand anti-mouse CD28 antibodies were treated with various concentrationsof BV-PLA2 (secretory type) for 3 days. The measured values werestandardized with regard to the values of the stimulated control group.(A) shows CD4⁺CD25⁺Foxp3⁺ Treg from control group and that from thesplenocytes treated with BV-PLA2 (0.1, 1 μg/ml). The numbers within thedot plots represent the percentage of cells belonging to thecorresponding quadrant (B) shows the percentages of CD4⁺CD25⁺Foxp3⁺Treg, which was significantly increased in the CD4⁺CD25⁺Foxp3⁺Treg-treated group compared to the control group. The data is indicatedvia mean±SEM (standard error of the mean), and the analysis of thestatistics was performed via Newman-Keuls multiple comparison testfollowing the one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001).

FIG. 11 shows graphs illustrating the effect of BV-PLA2 (secretory type)on cytokine production. The splenocytes stimulated with anti-mouse CD3antibodies and anti-mouse CD28 antibodies were treated with variousconcentrations of BV-PLA2 for 3 days. The supernatant of the resultingculture was recovered, centrifuged at 4° C. at 300 rcf for 10 minutes,and the resultant was stored at −70° C. for future use. The measuredvalues were standardized with regard to the values of the stimulatedcontrol group. Cytokines were measured via flow cytometry usingcytometric bead array (CBA). The data is indicated via mean±SEM, and theanalysis of the statistics was performed via Newman-Keuls multiplecomparison test following the one-way ANOVA (*p<0.05, **p<0.01,***p<0.001).

FIG. 12 shows CD4⁺CD25⁺Foxp3⁺ Treg populations in an ovalbumin(OVA)-induced asthma group, which are the results in, in a clockwisedirection from the left upper end, control group (CON); a group with notreatment after OVA induction (OVA); a group introduced to with BV-PLA2after OVA induction (OVA+PLA2); a group introduced with BV-PLA2(secretory type) after OVA induction and anti-CD25 antibodies injection(OVA-T+PLA2); and a group with OVA induction and introduction withanti-CD25 antibodies injection (OVA-T). Specifically, isolated alveolarcells were stained with anti-CD4 allophycocyanine and anti-CD25phycoerythrin (PE), and analyzed via flow cytometry. The data isindicated via mean±SEM, and the analysis of the statistics was performedvia Newman-Keuls multiple comparison test following the one-way ANOVA(n=4).

FIG. 13 shows graphs illustrating the effect of BV-PLA2 (secretory type)on the production of IL-4 and IL-13 cytokines in bronchoalveolar lavagefluid (BALF). The level of Th2 inflammatory cytokine was shown higher ingroups of OVA, OVA-T and (OVA-T+PLA2) than in groups of (OVA+PLA2) andCON. The data is indicated via mean±SEM, and the analysis of thestatistics was performed via Newman-Keuls multiple comparison testfollowing the one-way ANOVA (***p<0.001, **p<0.05 vs. CON, n=4).

FIG. 14 shows a graph illustrating the IgE level in the blood sera ofthe control group and the experimental groups. Blood samples werecollected via cardiac puncture and the level of IgE in the samples wasanalyzed via ELISA. The data is indicated via mean±SEM, and the analysisof the statistics was performed via Newman-Keuls multiple comparisontest following the one-way ANOVA (*p<0.05, **p<0.01 vs. CON, n=4).

FIG. 15 shows graphs illustrating the effect of BV-PLA2 (secretory type)on the recruitment of leucocytes in the airway of OVA-induced asthmamice. After establishing the OVA-induced asthma mouse model, BLAF wascollected from the lungs of the mice. The total cells, eosinophils,macrophages, neutrophils, and lymphocytes were counted from the BALF.The data is indicated via mean±SEM, and the analysis of the statisticswas performed via Newman-Keuls multiple comparison test following theone-way ANOVA (*p<0.05, **p<0.01, ***p<0.001 vs. CON and OVA, n=4).

FIG. 16A-E shows pictures illustrating the effect of BV-PLA2 (secretorytype) on the recruitment of inflammatory cells into the lung tissues ofOVA-induced allergic asthma mice. Balb/c Foxp3^(+EGFP) Balb/c mice weresensitized with OVA and test-infected. The lung tissues were stainedwith hematoxylin and eosin (H&E) (400× magnification). The results shownrepresent: (A) a group of PBS-test infected mice treated with PBS (CON);(B) a group of OVA-test infected mice treated with PBS (OVA); (C)OVA-test infected mice treated with BV-PLA2 (0.2 mg/kg) (OVA+PLA2); (D)a group of OVA and anti-CD25 antibodies-test infected mice treated withBV-PLA2 (0.2 mg/kg) (OVA-T+PLA2); and (E) a group of OVA and anti-CD25antibodies-test infected mice (OVA-T).

FIG. 17A-E shows pictures illustrating the effect of BV-PLA2 (secretorytype) on the histopathological changes in the lung tissues ofOVA-induced allergic asthma mice. The lung tissues were stained withperiodic acid-Schiff (PAS) (400× magnification). The results shownrepresent: (A) a group of PBS-test infected mice treated with PBS (CON);(B) a group of OVA-test infected mice treated with PBS (OVA); (C)OVA-test infected mice treated with BV-PLA2 (0.2 mg/kg) (OVA+PLA2); (D)a group of OVA and anti-CD25 antibodies-test infected mice treated withBV-PLA2 (0.2 mg/kg) (OVA-T+PLA2); and (E) a group of OVA and anti-CD25antibodies-test infected mice (OVA-T).

FIG. 18 shows a graph illustrating the effect of BV-PLA2 (secretorytype) on the hyperresponsiveness in an OVA-induced asthmatic mousemodel. The OVA-induced asthmatic mice had high P_(enh) value at 50 and100 mg/ml concentrations of methacholine (*p<0.01 vs. CON). The group(OVA+PLA2) show a significant decrease in the P_(enh) value at 100 mg/mlconcentration of methacholine used in the OVA-induced mice (regardingOVA, **p<0.01 vs. PLA2).

FIG. 19A-E shows a graph illustrating the preventive effect of BV-PLA2(secretory type) on dopaminergic (DA) neuronal cell death in substantianigra (SN) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-addicted mice. The MPTP-addicted mice were treated with BVPLA2(0.5 mg/kg) or brine for 6 days starting 12 hours after the final MPTPinjection. Some of the mice were administered with the anti-CD25antibodies (1 mg/kg) one day prior to the MPTP injection. Seven daysafter the MPTP-addiction, the brain slices were immunostained with α-THantibodies for the DA neurons. (A) shows the result where thebrine-injected mice were treated with brine, (B) shows the result wherethe MPTP-addicted mice were treated with brine, and (C) shows the resultwhere the MPTP-addicted mice were treated with BV-PLA2. (D) and (E) showthe number of TH positive neurons counted. For each experimental group,8 to 9 experimental animals were used. In (A) through (C), the scale barrepresents a length of 200 μm (*p<0.001 vs. brine-treated control group,#p<0.01 vs. MPTP-addicted mice).

FIG. 20A-B: (A) shows a vector structure for the expression of arecombinant BV-PLA2, and (B) shows the amino acid constitutions of fourtypes of recombinant BV-PLA2.

FIG. 21A-B: (A) shows an SDS-PAGE for confirmation of the recombinantproteins produced, and (B) shows the result of activity analysis foreach enzyme.

FIG. 22A through 22E show the results of flow cytometry analysis of thenaturally occurring and recombinant BV-PLA2s stained with anti-CD4 andanti-CD25 antibodies for confirmation of their effects on theCD4⁺CD25⁺Foxp3⁺ regulatory T cells.

FIG. 23 is a graph showing the effects of the naturally occurring andrecombinant BV-PLA2s on Foxp3+ population among the CD4⁺ cells.

FIG. 24A-B: (A) shows a result of SDS-PAGE for confirmation of theC-type recombinant BV-PLA2 and C-type mutant (H34Q) recombinant BV-PLA2,and (B) shows a comparison result of the activities of a naturalsecretory BV-PLA2 and the above enzymes.

FIG. 25A-B shows (A) a graph showing the effects of the naturalsecretory BV-PLA2, C-type recombinant BV-PLA2, and C-type mutant (H34Q)recombinant BV-PLA2 on the Foxp3⁺ population among splenocytes, and (B)a graph showing the effects of the natural secretory BV-PLA2, C-typerecombinant BV-PLA2, and C-type mutant (H34Q) recombinant BV-PLA2 on theFoxp3⁺ population among CD4⁺ cells.

BEST MODE

In an aspect to accomplish the above objectives, the present inventionprovides a pharmaceutical composition, for treating or preventingdiseases related to abnormal suppression of regulatory T cell (Treg)activity, containing a polypeptide including a BV-PLA2 amino acidsequence exclusive of a leader sequence as an active ingredient.

As used herein, the term “bee venom (BV)” refers to a mixture of acidicand basic secretions generated in the abdomen of a honey bee (Apismellifera) in the form of a colorless liquid, which includes as maincomponents peptides such as melittin, apamin, and mast celldegranulating (MCD) peptide and phospholipase A2 (PLA2), etc., and atrace amount of various components. As used herein, the term “beevenom-phospholipase A2 (BV-PLA2)” refers to phospholipase A2 among thevarious complex mixtures of bee venom. As used herein, the term“phospholipase A2 (PLA2) refers to an enzyme which serves the functionof fatty acids via hydrolysis of glycerol at the 2^(nd) carbon position.It specifically recognizes an sn-2 acyl bond of phospholipids andcatalyzes hydrolytic activity thereby releasing arachidonic acid andlysophospholipids. Generally, PLA is not only found in bacteria, insectsor serpent venoms but also in the tissues of mammals. The BV-PLA2 of thepresent invention may be derived from a honey bee (Apis mellifera) butis not limited thereto.

BV-PLA2 may not be particularly limited as long as it has the PLA2function derived from bee venom, and in particular, may be composed of asequence represented by SEQ ID NO: 1. The SEQ ID NO: 1 is the totalamino acid sequence that can be expressed from a gene regarding thepublicly known BV-PLA2 (GenBank ID: ABQ28728.1), and is in the form ofactual secretion. That is, it is the secretory type of BV-PLA2 in theform exclusive of a leader sequence at the N-terminus, and specifically,it may have an amino acid sequence represented by SEQ ID NO: 2(consisting of 34th to 167 amino acids of SEQ ID NO: 1). As used herein,the term “leader sequence” refers to a sequence which is removed duringthe maturation process of from BV-PLA2 to a secretory bee venom-PLA2,and it may be a sequence corresponding to from the 1^(st) to 33^(rd)amino acids, and specifically, a sequence represented by SEQ ID NO: 3.In the present invention, the BV-PLA2 exclusive of the leader sequenceis used as the same as the secretory BV-PLA2. The BV-PLA2 exclusive ofthe leader sequence is also called a matured type.

In the present invention, the polypeptide including a BV-PLA2 amino acidsequence exclusive of a leader sequence may be one having an additionalamino acid sequence on C-terminal region and/or N-terminal region of theBV-PLA2 amino acid sequence exclusive of a leader sequence.Specifically, the polypeptide may be one having an additional amino acidsequence represented by SEQ ID NO: 4 on the N-terminal region, or onehaving an additional amino acid sequence represented by SEQ ID NO: 5 onthe C-terminal region. More specifically, the polypeptide may be onehaving an amino acid sequence represented by SEQ ID NO: 6. The aboveadditional amino acid sequences may include a tag for purifyingrecombinant proteins.

The polypeptide of the present invention including the bee venom-PLA2amino acid sequence exclusive of a leader sequence, may be prepared byseparating from bee venom by a known method, a recombinant expression,or may be used after purchasing those available in the market, orprepared by synthesis, but is not limited thereto.

In an exemplary embodiment of the present invention, it was confirmedthat a C-type recombinant BV-PLA2 (SEQ ID NO: 6; FIG. 20B), which wasprepared by connecting additional amino acid residues for purificationto both termini of the secretory type naturally-occurring BV-PLA2 aminoacid sequence, exhibited an activity equivalent to or improved than thatof the naturally-occurring BV-PLA2 (FIGS. 21B and 23). In contrast, anF-type recombinant BV-PLA2, which was prepared by including additionalleader sequence to the N-terminus of the secretory typenaturally-occurring BV-PLA2 amino acid sequence, or a B-type recombinantBV-PLA2, which was prepared by including additional leader sequence tothe N-terminus and cutting off a part of the C-terminus (SEQ ID NOS: 7and 8; FIG. 20B) failed to exhibit any significant enzyme activities(FIGS. 21B and 23).

The BV-PLA2 of the present invention is a protein with a molecularweight of about from 10 kDa to 18 kDa, whose constitution is differentfrom other PLA2s derived from other biological organisms or tissues, inparticular, has only 9% of sequence homology to that of human PLA2(based on the amino acid sequence of SEQ ID NO: 2).

The BV PLA2 amino acid sequence not only includes a naturally-occurringamino acid sequence but also includes its sequence derivative (mutein)or its fragment exhibiting the BV-PLA2 activity. As used herein, theterm “amino acid sequence derivative” refers to an amino acid sequencewhich differs from the naturally-occurring amino acid sequence bydeletion, insertion, non-conservative substitution, or conservativesubstitution or a combination thereof by at least one amino acid residuein the naturally-occurring amino acid sequence.

In an exemplary embodiment of the present invention, it was observedthat the C-type mutant (H34Q) recombinant BV-PLA2, which was prepared bysubstituting histidine, the 34^(th) amino acid from the N-terminus ofthe secretory naturally-occurring BV-PLA2, into glutamine, showed adrastic decrease in its activity (FIGS. 24 and 25). Accordingly, it ispreferred that at least histidine, the 34^(th) amino acid, be includedas an essential component for enzyme activity.

In the present invention, its fragment exhibiting the BV-PLA2 activitymay have an amino acid sequence of SEQ ID NO: 9. A polypeptide includingan amino acid sequence of a BV-PLA2 fragment exclusive of a leadersequence may be a polypeptide which further includes an additional aminoacid sequence to the C-terminus or N-terminus of the amino acid sequenceof BV-PLA2 fragment exclusive of a leader sequence. Specifically, it maybe a polypeptide additionally including an amino acid sequence of SEQ IDNO: 4 to an N-terminus region, and a polypeptide additionally includingan amino acid sequence of SEQ ID NO: 5 to a C-terminus region. Morespecifically, it may be a polypeptide including an amino acid sequenceof SEQ ID NO: 10. The above additional amino acid sequence may include atag for the purification of recombinant proteins.

The substitution of amino acids in proteins or peptides withoutmodifying the entire molecular activity is already known in the art (H.Neurath and R L. Hill, The Proteins, Academic Press, New York, 1979).The most conventional substitution is the substitution between aminoacid residues of Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly. Depending on the situations, thesubstitution may be modified via phosphorylation, sulfation, acrylation,glycosylation, methylation, famesylation, acetylation, amidation, etc.

The above described sequence derivatives may be those which exhibit thesame biological activity to BV-PLA2 of the present invention or have animproved structural stability regarding heat, pH, etc.

As used herein, the term “regulatory T cell (Treg)” refers to a kind ofhelper T cells (Th) also called suppressor T cells. Unlike other helperT cells exhibiting a humoral immune promoting effect by acceleratingdifferentiation and activation of other leucocytes, Treg maintainsimmune tolerance by inhibiting immunity thereby enabling homeostasis.Treg can be divided into adaptive Treg, which includes Tr1 and Th3 cellsproduced during normal immune responses, and naturally-occurring Treg.The naturally-occurring Treg is generated in thymus, has positive tophenotypes for CD4, CD25 and Foxp3, and is distinguished from other Tcells due to the presence of Foxp3, an intracellular molecule.

As used herein, the term “diseases related to abnormal suppression ofTreg activity” refers to a disease that occurs when the activity ofregulatory T cells, which performs the functions of inhibition ofautoimmunity, immune tolerance, inhibition of tissue damage byinflammation, etc., becomes abnormally deteriorated, and it includesallergic diseases, autoimmune diseases and neurodegenerative diseases,but is not limited thereto.

The distinction between self and non-self antigens is the most importantissue in the field of immunology, and due to the studies for the pastfew decades, various mechanisms involved in central tolerance andperipheral tolerance maintaining unresponsiveness to self antigens havebeen identified. In addition to the mechanisms of clonal deletion andanergy (functional inactivation), the suppression of the activity ofself-reactive T cells has been also considered as an important mechanismin maintaining the tolerance for self-antigens.

Among them, CD4⁺CD25⁺ T cells, known as naturally-occurring Tregs, havebeen confirmed to be important for the tolerance and prevention ofautoimmune diseases by the experimental result that they can inducevarious kinds of autoimmune diseases in mice where CD25⁺ cell deficientT cells or CD25⁻ T cells were introduced via adoptive transfer (S.Sakaguchi, Annu. Rev. Immunol., 2004, 22: 531-562), before the discoverythat foxp3 is an essential regulator gene important for Treg cellinduction.

It is known that foxp3 mutants can induce X-linked immunodeficiencysyndrome (IPEX), which directly induces autoimmune diseases (type Idiabetes, thyroiditis, etc.) from many organs in the endocrine system inhumans (C. L. Bennett et al., Nat. Genet., 2001, 27: 20-21; R S. Wildinet al., Nat. Genet., 2001, 27: 18-20). This suggests the importance ofCD4⁺CD25⁺Foxp3⁺ regulatory T cells in tolerance and for the preventionof autoimmune diseases.

In addition to IPEX, the deficiency in Tregs or the loss of theirsuppressive function have been found to be associated with thepathogenic mechanism of autoimmune diseases such as type I diabetes,rheumatoid arthritis, multiple sclerosis, and psoriasis (M. Miyara etal., J. Immunol., 2005, 175: 8392-8400).

Attempts have been made to use self antigen-specific Tregs for thetreatment of autoimmune diseases and prevention of graft rejection.These attempts are mostly in the form of a somatic cell therapy, whereinthe amplification of self antigen-specific Tregs is induced in vivo asin the case of peptide therapeutic vaccination using altered peptideligand (APL) or administered after in vitro amplification, and aremethods that inhibit the functions of effector T cells, which arespecific to self antigens that induce autoimmunity (Y. Belkaid and B. T.Rouse, Nat. Immunol., 2005, 6: 353-360).

Although pathogen-specific Tregs have a negative impact on theelimination of pathogens they serve an important role in the regulationof immunopathology by inhibiting tissue damage due to inflammationinduced by pathogens, as in the prevention of autoimmune diseases (YBelkaid and B. T. Rouse, Nat. Immunol., 2005, 6: 353-360).

For example, in an experiment using mice infected with HSV throughfoodpads, deficiency in CD4⁺CD25⁺ T cells improved virus elimination byincreasing CD8 but it aggravated T cell-mediated lesions (S. Suvas etal., J. Exp. Med., 2003, 198: 889-901).

This has become an important issue to consider when conducting atherapeutic vaccination for the treatment of chronic virus infectionsincluding HIV and HCV by targeting Tregs such as removal ofnaturally-occurring T cells or blocking of effector molecules (Y Belkaidand B. T. Rouse, Nat. Immunol., 2005, 6: 353-360).

As used herein, the term “autoimmune diseases” may include diseases thatdestroy self materials by wrongly determining self materials as foreignmaterials due to the inability to distinguish self materials frommaterials introduced from outside, which is the most important step inimmune responses in the course of a series of actions for protectingself by destroying or inactivating materials introduced from outside.For example, they made include rheumatoid arthritis, systemic sclerosis,insulin-dependent juvenile diabetes by pancreatic islet cell antibody,alopecia areata, psoriasis, pemphigus, asthma, aphthous stomatitis,chronic thyroiditis, partial acquired aplastic anemia, primary livercirrhosis, ulcerative colitis, Behcet's disease, Crohn's disease,silicosis, asbestosis, IgA nephropathy, poststreptococcalglomerulonephritis, Sjogren syndrome, Guillian Barre syndrome,dermatomyositis, polymyositis, multiple sclerosis, autoimmune hemolyticanemia, autoimmune encephalomyelitis, myasthenia gravis, Graves disease,polyarteritis nodosa, ankylosing spondylitis, fibromyalgia, temporalarteritis, Wilson's disease, Fanconi syndrome, multiple myeloma, andsystemic lupus erythematosus, but is not limited thereto. The aboveautoimmune disease may be multiple sclerosis or autoimmuneencephalomyelitis

In an exemplary embodiment of the present invention, it was observedthat bee venom inhibited the differentiation of Th1/Th17 therebyalleviating experimental autoimmune encephalomyelitis (EAE) (FIG. 5)while increasing the expression of Foxp3 in CD4⁺CD25⁺Foxp3⁺Treg andCD4⁺CD25⁺ Treg, which are regulatory T cells (FIG. 2) involved inimmunities. Additionally, it was confirmed that Treg-deficiency canblock the EAE alleviating effect of bee venom (FIG. 6), and thussuggested that Treg acts as an important mediator of EAE alleviation bybee venom. Accordingly, the increase of CD4⁺CD25⁺Foxp3⁺ Treg anddecrease of Th1/Th17 by bee venom in human CD4⁺ T cells were confirmedthereby verifying the immune regulation effect of bee venom in humans.Additionally, upon measurement of immune regulation inducing effects ofvarious bee venom components, PLA2 (secretory type) was shown to havethe highest immune regulation effect among the bee venom components(FIG. 8), and by its application in human cells, it was confirmed toenable induction of immune regulation in humans (FIG. 9).

As used herein, the term “allergic diseases” refers to diseases, whereinsubstances which are non-toxic to normal people cause various symptomsin particular people due to abnormalities in their systems. Thesubstances causing the allergic diseases are called allergens orantigens, and pollens, antibiotics, drugs, dusts, foods, cold air orsunlight, etc., may induce allergies. Examples of the allergic diseasesinclude hives, sneezing, rhinorrhea, coughs, hay fever, ocularhyperemia, eczema, rashes, etc. The symptoms of the allergic diseasesare hives, sneezing, pruritus, rhinorrhea, coughs, hay fever, ocularhyperemia, eczema, rashes, etc. The allergic diseases may includeasthma, atopic dermatitis, conjunctivitis, rhinitis, and ulcerativecolitis, but is not limited thereto. Preferably, the allergic disease isasthma, which accompanies symptoms such as airway constriction, increasein the secretion of mucus liquid in the lungs, dyspnea, and coughs.

In an exemplary embodiment of the present invention, when an OVA-inducedasthma mouse model, where the asthma was induced by sensitization withovalbumin (OVA), was treated with BV-PLA2 there was a significantincrease in CD4⁺CD25⁺Foxp3⁺ Treg compared to the control group and otherexperimental groups (FIG. 12), thus confirming that BV-PLA2 has anexcellent inhibitory effect against the introduction of inflammatorycells into lung tissues increased due to OVA sensitization (FIG. 16).Additionally, it also showed an effect of alleviating the airwayhyperresponsiveness (AHR) (FIG. 18).

As used herein, the term “neurodegenerative diseases” refers to diseasesassociated with symptoms such as degeneration, loss of functions, andoften apoptosis of neurons. Since these symptoms are mostly progressivethey are often highly destructive and patients with neurodegenerativediseases may experience extreme deterioration in their cognitive ormotor performance. The neurodegenerative diseases may include, althoughnot particularly limited thereto, Parkinson's disease (PD), Alzheimer'sdisease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease(HD), fronto-temporal dementia, cortico basal degeneration, andprogressive to supranuclear palsy (PSP).

In an exemplary embodiment of the present invention, it was confirmedthat BV-PLA2 (secretory type) decreases dopaminergic (DA) neuronal deathin substantia nigra (SN) of a1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-addicted mouse,i.e., an animal model with Parkinson's disease (FIG. 19).

As used herein, the term “prevention” refers to all kinds of activitiesof inhibiting diseases related to abnormal suppression of regulatory Tcell activity or delaying their occurrence by administering apharmaceutical composition of the present invention, and the term“treatment” refers to all kinds of activities of improving oradvantageously modifying the symptoms of the diseases related toabnormal suppression of regulatory T cell activity by administering thepharmaceutical composition of the present invention.

The pharmaceutical composition of the present invention may furtherinclude a pharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable” refers to a non-toxic property to cells orhumans being exposed to the composition. The above carrier to be usedmay be any one known in the art such as a buffering agent, apreservative, a pain-relieving agent, a solubilizing agent, an isotonicagent, a stabilizing agent, a base, an excipient, a lubricant, etc.,without any limitation. The carrier, excipient, and diluent that may beincluded in the pharmaceutical composition of the present invention arelactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate,calcium silicate, cellulose, methyl cellulose, microcrystallinecellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and base oil. For thepreparation of formulations, the conventionally used filler, extender,binder, humectants, disintegrating agent, surfactant, diluent orexcipient may be used. Examples of the usable typical surfactant thatmediates the transmembrane transport are those derived from steroids,cationic lipids such asN-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride] (DOTMA), orcholesterol hemisuccinate.

In another aspect of the present invention, there is provided a methodfor preventing or treating diseases related to abnormal suppression ofregulatory T cell activity including administering the abovepharmaceutical composition to a subject in need thereof.

As used herein, the term “subject” refers to all kinds of animalsincluding humans in which diseases related to abnormal suppression ofregulatory T cell activity have occurred or may occur, and the diseasesrelated to abnormal suppression of regulatory T cell activity can beeffectively prevented or treated by administering the pharmaceuticalcomposition of the present invention to the subject. The pharmaceuticalcomposition of the present invention may be administered in combinationwith a conventional therapeutic agent for the diseases related toabnormal suppression of regulatory T cell activity.

As used herein, the term “administration” refers to an introduction of aparticular material to a subject by a suitable method, and the abovecomposition may be administered via any conventional administrationroute as long as they can allow the composition to arrive at the targettissue. For example, intraperitoneal administration, intravenousadministration, intramuscular administration, intradermaladministration, oral administration, topical administration, intranasaladministration, intranasal administration, intrapulmonaryadministration, and intrarectal administration, but is not limitedthereto. Solid formulations for oral administration may include tablets,pills, powders, granules, capsules, etc., and these solid formulationsare prepared by adding at least one excipient in addition to thecomposition, for example, starch, calcium carbonate, sucrose, orlactose, gelatin, etc. Additionally, a lubricant such as magnesiumstearate, and talc may be used, in addition to a simple excipient.Examples of liquid formulations for oral administration may includesuspensions, medicines for internal use, emulsifiers, syrups, etc., andin addition to the frequently used simple diluents such as water andliquid paraffin, various excipients, for example, humectants,sweeteners, fragrant, preservatives, etc., may be included. However,peptides can be easily digested if administered orally, and thus acomposition for oral administration is preferred to be formulated insuch a manner that the active drug component is coated or protected fromdecomposition in the stomach. Formulations for parenteral administrationmay include sterile aqueous solutions, nonaqueous solvents, suspensions,emulsifiers, lyophilized preparations, suppositories. Examples of thenonaqueous solvents and suspensions include propylene glycol,polyethylene glycol, a vegetable oil such as olive oil, an injectableester such as ethyl oleate. Examples of a base for the suppositoriesinclude witepsol, macrogol, Tween 61, cacao butter, laurinum,glycerogelatin, etc. For the improvement of stability or absorptivity ofpeptides, carbohydrates such as glucose, sucrose, and dextran;antioxidants such as ascorbic acid and glutathione; chelating materials,low molecular weight proteins or other stabilizers may be used.

Additionally, the pharmaceutical composition of the present inventionmay be administered by any device which enables an active ingredient tomove to a target cell. Preferable administration routes and formulationsinclude those for intravenous injection, subcutaneous injection,intradermal injection, intramuscular injection, intravenous infusion,etc. Injections may be prepared using an aqueous solvent such as saline,ringer solution, etc.; a non-aqueous solvent such as a vegetable oil,high grade fatty acid eser (e.g., oleic acid ethyl, etc.), alcohols(e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), etc.,and may include a pharmaceutically acceptable carrier such as astabilizer for preventing deterioration (e.g., ascorbic acid, sodiumbisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), anemulsifier, a buffer for pH adjustment, a preservative for preventingmicrobial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkoniumchloride, phenol, cresol, benzyl alcohol, etc.).

Meanwhile, the pharmaceutical composition of the present invention isadministered in a pharmaceutically effective amount. As used herein, theterm “pharmaceutically effective amount” refers to an amount sufficientfor the treatment of diseases at a reasonable benefit/risk ratioapplicable to a medical treatment without causing any adverse effects,and the level of an effective dose may be easily determined by a skilledperson in the art according to factors including sex, age, body weight,health status of a patient, type of disease(s), severity of illness,drug activities, drug sensitivity, route and duration of administration,release rate, treatment period, mixing of drugs or other drug(s) used incombination, and others well known in the medical field. In general, anactive ingredient may be administered daily in the amount of about from0.01 mg/kg/day to 1000 mg/kg/day. For oral administration, it may beappropriate to administer in the amount of from 50 to 500 mg/kg, and maybe administered at least once daily.

MODE FOR INVENTION

Hereinafter, the present invention will be described in more detail withreference to the following examples. However, the following examples areprovided for illustrative purposes only, and the scope of the presentinvention should not be limited thereto in any manner.

Example 1: Preparation of Experimental Animals

Seven to eight-week old female C57BL/6 mice with a body weight of from18 g to 23 g were purchased from Charles River Korea (Sungnam, Korea).Foxp3^(EGFP)C57BL/6 (C. Cg-Foxp3tm2Tch/J) mice were purchased fromJackson Laboratory (Bar Harbor, Me., USA) and seven to eight week oldmale C57BL/6J mice with a body weight of from 21 g to 22 g werepurchased from Charles River Breeding Laboratory (Yokohama, Japan). Allmice were stored under aseptic conditions provided with air conditioningand 12 hour light/dark cycles. Additionally, all mice were given adlibitum access to food and water during the experiment. This study wasapproved by Animal Experimentation Ethics Committee of Kyung HeeUniversity (Korea).

1.1. Experimental Autoimmune Encephalomyelitis (EAE)-InducedExperimental Animal Model

Murine MOG₃₅₋₅₅ (M-E-V-G-W-Y-R-S-P-F-S-R-V-V-H-L-Y-R-N-G-K) (SEQ IDNO:24) peptides were purchased from Peptron Inc. (Daejeon, Korea). Thepeptides were purified via HPLC to ensure 95% or higher of purity. ForEAE induction, 200 μg of MOG₃₅₋₅₅ peptides, which was dissolved in CFA(Sigma-Aldrich, St. Louis, Mo., USA) containing 100 μg of Mycobacteriumtuberculosis, were administered to the hind flanks of mice viasubcutaneous injection, and further administered with 400 ng ofpertussis toxin (Sigma-Aldrich, St. Louis, Mo., USA) via intraperitonealinjection on day 0 and day 2. The mice were examined daily to check thepresence of clinical signs of any disease(s), and the results werescaled in the range of from 0 to 5 at 0.5 intervals; wherein 0 denotesno clinical sign, 1 denotes relaxed tails, 2 denotes weakened hind legsor abnormal gait, 3 denotes complete paralysis of hind legs, 4 denotescomplete paralysis of hind legs accompanying weakened front legs orparalysis of front legs, and 5 denotes being in a moribund state ordeath.

1.2. Allergic Asthma Experimental Animal Model

Six to eight-week old female Foxp3^(+EGFP) Balb/c mice were prepared.The mice on day 0 were divided into a negative control group(PBS-treatment; CON) (n=4) and an OVA-induced asthma group. TheOVA-induced asthma group was subdivided into four groups(OVA-test-infected mice with PBS treatment (OVA); OVA-test-infected micewith BV-PLA2 (0.2 mg/kg) treatment (OVA+PLA2); OVA and anti-CD25antibody-test-infected mice with BV-PLA2 (0.2 mg/kg) treatment(OVA-T+PLA2); and OVA and anti-CD25 antibody-test-infected mice (OVA-T),n=4/group). Briefly, the mice were sensitized via intraperitoneal (ip)injection of 100 μg of ovalbumin (OVA; Sigma-Aldrich, St. Louis, Mo.,USA), which was precipitated with 20 mg aluminum hydroxide in 100 μl ofPBS on day 0 and day 14. The mice were test-infected by directlyadministering 1% OVA in 50 μl of PBS through their nostrils using amicropipette through a total of 6 administrations from day 20 to day 30.The mice in the negative control group were sensitized with PBS aloneand test-infected.

Treg-deficient mice were intraperitoneally injected with 0.25 mganti-CD25 antibodies via on days 1, 8 and 15. Anti-mouse CD25 rat IgG1(anti-CD25; clone PC61) was internally manufactured from hybridomasobtained from American Type Culture Collection (Manassas, Va., USA). Theefficacy of Treg-deficiency as confirmed via flow cytometry analysisusing PE-anti-mouse CD25 and fluorescein isothiocyanate(FITC)-anti-mouse CD4. The mice in the PLA2-test infected group wereintraperitoneally injected with PLA2 at a concentration of 0.2 mg/kgthrough a total of 6 injections from day 3 to day 17. On day 31, airwayhyperresponsiveness (AHR) was analyzed via (non-invasive) lung functionmeasurement (All Medicus, Seoul, Korea), and on day 32 mice weresacrificed and various tissues were collected therefrom for analyses(FIG. 1).

1.3. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-AddictedParkinson's Disease Experimental Animal Model

Seven to eight-week old male C57BL/6 mice with a body weight of from 21g to 22 g (Charles River Breeding Laboratory, Yokohama, Japan) wereprepared. For the construction of an MPTP-addicted model, the mice wereintraperitoneally injected with an MPTP-HCl (a free base brine at 20mg/kg; Sigma-Aldrich, St. Louis, Mo., USA) four times at two hourintervals. Twelve hours after the final MPTP injection, theMPTP-addicted mice were intraperitoneally injected with BV-PLA2 (0.5mg/kg) once daily for six days. Some of the mice used as a control groupwere injected with BV-PLA2 alone or a vehicle. For Treg deficiencypurpose, some of the mice were intraperitoneally injected with anti-CD25antibodies (1 mg/kg) or normal rat IgG (1 mg/kg) once one day prior tothe administration of MPTP

Example 2: Separation of Human Peripheral Blood Mononuclear Cells (PBMC)

Human PBMC was separated from a healthy donor's blood collected using aBD VACUTAINER® CPT tube containing heparin sodium. Briefly, the bloodwas directly collected into the tube via venipuncture and centrifuged at1600 g for 20 minutes. The cells located on the upper portion of thecentrifuged gel were recovered using a pipette and washed twice withPBS. The present invention was approved by the Institutional ReviewBoard (IRB) of Korean Medicine Center of Kyung Hee University (Korea).

Example 3: Separation of Murine T Cells

CD4⁺, CD4⁺CD25⁻ T cells and CD4⁺CD25+ Treg were separated from thesplenocytes obtained from female Foxp3^(EGFP)C57BL/6 mice via magneticbead separation (CD4⁺ T cell separation kit and CD4⁺CD25⁺ regulatory Tcell kit; Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany) accordingto the manufacturer's instructions. The purities of all the populationswere determined via flow cytometry analysis, and they conventionallyreached 90% or higher.

Example 4: Reagents and Administration Routes

Bee venom (BV), melittin, secretory bee venom-derived phospholipase A2(BV-PLA2), mast cell degranulating peptide (MCD) and apamin wereprepared in PBS solutions for in vitro experiments. Each component wasused according to the proportion of the venom, that is, 50% of melittin,10% of BV-PLA2, 2% of apamin, and 1% of MCD. Bee venom and allcomponents of bee venom were purchased from Sigma-Aldrich (St. Louis,Mo., USA). Bee venom stock solution was prepared by dissolving bee venomin 0.9% brine at a concentration of 1 mg/ml. The bee venom stocksolution was used after dilution according to concentrations fordose-dependent in vivo experiments. Bee venom (0.01, 0.1, 1, and 10mg/kg body weight) or an equal volume of brine was intraperitoneallyinjected into the mice daily.

Example 5: Growth Analysis

In an EAE-induced mouse model, the murine splenocytes immunized withMOG₃₅₋₅₅ peptides were separated when the symptoms of the diseasereached its culminating point. Briefly, the splenocytes were cultured ata concentration of 2×10⁶/ml in RPM 1640, which was added with 10% FBS,50 IU/ml of penicillin, 50 μg/ml of streptomycin, 10 μg/ml of MOG₃₅₋₅₅peptide and bee venom (0.01, 0.1, 1, and 10 μg/ml), for 72 hours. Inorder to determine the inhibitory effect of BV against antigen-specificgrowth, the number of splenocytes was counted using a hemocytometer. Allthe experiments were repeated three times.

Example 6: Flow Cytometry Analysis

All the cytometry analyses regarding the samples prepared according tothe methods described below were performed using CellQuest Pro Software(BD Biosciences, San Jose, Calif., USA) after collecting data using FASCCalibur flow cytometer (BD Bioscience, San Jose, Calif., USA).

6.1. Cell Preparation for Cytometry Analysis in EAE-Induced Animal ModelExperiments

In order to define Treg, a suspension of separated single-cells obtainedfrom Foxp3^(EGFP)C57BL/6 mice and splenocytes, and a healthy donor'sPBMC were labeled with anti-CD4 and anti-CD25 or anti-Foxp3 monoclonalantibodies (eBioscience, San Diego, Calif., USA) according to thestandard staining method. For the detection of intracellular cytokineexpressions in mice and human cells, intracellular staining wasperformed using a cytofix/cytoperm kit (BD Biosciences, San Jose,Calif., USA) according to the manufacturer's instructions. Briefly, thecells were restimulated with 50 ng/ml of PMA and 1 μg/ml of ionomycin(both from Sigma-Aldrich, St. Louis, Mo., USA) for 5 hours in thepresence of BD GOLGISTOP™ protein transport inhibitor. The cells werestained with BD mouse or human Th1/Th2/Th17 phenotyping cocktailaccording to the manufacturer's instructions. For the detection ofcytokine production, the secreted cytokines were measured usingcytometric bead array (CBA; BD Biosciences, San Jose, Calif., USA)according to the manufacturer's instructions. Twenty four days afterimmunization, sera were collected and stored at −20° C. before use forCBA analyses.

6.2. Cell Preparation for Cytometry Analysis in Allergic Asthma-InducedAnimal Model Experiments

After separating CD4⁺ cells from the spleen of Foxp3^(EGFP) Balb/c mice,they were seeded into a round bottom 96-well plate coated with anti-CD3e(2.5 μg/ml) for one day at a concentration of 4×10⁶ cells/ml in theamount of 200 μl/well, costimulated with anti-CD28 (2 μg/ml), added withBV-PLA2 at a concentration of 0.1, 1 μg/ml, and cultured in an incubator(37° C., 5% CO₂) for three days. The resulting liquid culture was storedfor cytokine measurement. Then, the resultant was added with CD4 andCD25 antibodies (anti-CD4-APC and anti-CD25-PE mAbs), which are cellsurface markers, and placed at 4° C. for 30 minutes. The resultant wascentrifuged at 300×g after suspending in 1 ml of Dulbecco's phosphatebuffered saline (DPBS) (welGENE) and the supernatant was removed. Theresultant was resuspended in 300 μl of DPBS, and the amount ofexpression of CD4⁺CD25⁺ Treg and Foxp3 were measured.

Additionally, for the analysis of cells in lung tissues, the lung wasisolated and washed with PBS to remove blood. The lung tissues were cutinto small pieces and lysed in a 1% RBC lysis buffer (BD Pharmingen).Upon lysis of RBC, the lung tissues were passed through a 25 μm cellstrainer. The cells were washed three times and resuspended in FCMbuffer solution (PBS containing 2% FBS and 0.1% NaN₃). The suspension ofthe single-cells obtained from FOXP3^(EGFP) Balb/c mice and the lungcells were labeled with anti-CD4-APC and anti-CD25-PE monoclonalantibodies according to the standard staining method, and the percentageof the cells stained with a particular sample was analyzed via FACSCalibur using CellQuest Software (BD Biosciences).

Example 7: Measurement of Cytokines in the Supernatant of Liquid Culture

In an allergic asthma-induced model, splenocytes stimulated withanti-mouse CD3 antibodies and anti-mouse CD28 antibodies were treatedwith various concentrations of BV-PLA2 for three days. The supernatantof the liquid culture was recovered and centrifuged at 4° C. at a rateof 300 rcf for 10 minutes, and stored at −70° C. for future use. Thelevel of measured cytokines was standardized relative to the cytokinelevel of the stimulated control group. Cytokines were measured viamurine cytometric bead array (CBA, BD Biosciences, San Jose, Calif.,USA). Briefly, 50 μl of a sample or standard sample at a concentrationof a base (0-5000 pg/ml) was added into 50 μl of a mixture of a captureantibody bead solution and a detection antibody phycoeiythrin (PE)sample. Then, the resulting mixture was incubated at room temperaturefor two hours without sunlight, and washed to remove the unbounddetection antibody PE sample. The data were collected using a flowcytometer (FACS Calibur, BD Biosciences Corp., San Jose, Calif., USA)and analyzed by a computer (CBA Software 1.1; BD Biosciences Corp.).

Example 8: Bronchoalveolar Lavage (BAL)

In an allergic asthma-induced model, BAL was collected by infusion andextraction of 1 ml of ice-cold PBS. The above procedure was repeated 3times and the lavage was collected (average volume of 2.0 ml). Therecovered BAL (70-80%) was centrifuged at 13000 rpm for 10 minutes. Cellprecipitate was resuspended in 1 ml of PBS and attached to a glass slidevia cytocnetrifugation. The total number of viable cells was measuredusing a hemocytometer according to trypan blue exclusion. Thedifferential counts on eosinophils, neutrophils, lymphocytes, andmacrophages were determined based on smear samples of bronchoalveolarlavage fluid (BALF) samples (5×10⁵ cells/200 μl) from an individualmouse stained with Diff-Quick (Life Technologies, Auckland, New Zealand)after counting 500 cells. Then, BALF was centrifuged and the supernatantto was stored at −70° C. The result was expressed as the total cellnumber×10⁴.

Example 9: Analysis of Th2 Cytokine in BALF Via Enzyme-LinkedImmunosorbent Assay (ELISA)

In an allergic asthma-induced model, the concentrations of Th2 cytokineIL-4 and IL-13 were measured using a quantitative sandwich ELISA kit(BD, San Diego, USA for IL-4 and R&D, Minneapolis, USA for IL-13). A96-well microplate was incubated at 4° C. overnight in a coating buffersolution along with anti-mouse IL-4 and IL-13 monoclonal antibodies,washed with PBS containing 0.05% Tween 20 (Sigma, MO, USA), and blockedrespectively with PBS containing 5% FBS at 4° C. for one hour and withPBS containing 1% BSA for one hour. Then, 100 μl of BALF was addedthereto and incubated at room temperature for two hours. Secondaryperoxidase was labeled in an assay diluent with biotinylated anti-mouseIL-4 and IL-13 monoclonal antibodies for one hour. Finally, the platewas treated with TMB base solution (KPL, San Diego, USA) for 30 minutes,and then added with 50 n1 of TMB stop solution to each well to stop thereaction. The absorbance at 450 nm was measured via a microplate reader(SOFT max PRO, version 3.1 Software, CA, USA). Here, the detection limitfor IL-4 and IL-13 ELISA was 500 ng/ml and 100 ng/ml, respectively.

Example 10: Measurement of Serum IgE Titer Via ELISA

In an allergic asthma-induced model, the mice were anesthetized withether 32 days after inducing the disease, and blood samples werecollected from the retro-orbital plexus of the mice. Sera samples wereobtained via centrifugation and stored at −20° C. until analysis.Regarding the sera, a 96-well immune microplate (Costar, NY, USA) wascoated with anti-mouse IgE monoclonal antibodies. The sera were dilutedwith PBS (assay diluents) containing 5% FBS at 1:250 ratio. The IgEmeasurement (BD Pharmingen) was used for a standardized sandwich ELISAaccording to the manufacturer's protocol. The absorbance at 450 nm wasmeasured via a microplate reader (SOFT max PRO, version 3.1 Software,CA, USA). Here, the detection limit for IgE ELISA was 100 ng/ml,respectively.

Example 11: Histological Examination

In an allergic asthma-induced model, trachea and lung tissues wereremoved from the mice. First, they were fixed with 4% paraformaldehyde,sunk into paraffin after dehydration, cut into 6 t m slices, and stainedwith Hematoxylin & Eosin (H&E) and Periodic Acid Schiff (PAS) reagents.

Example 12: Measurement of Airway Hyperresponsiveness (AHR) onMethacholine

In an allergic asthma-induced model, on the day immediately followingthe final test-infection by administration of 50 μl of PBS containing 1%OVA, the experimental animals were put into a barometricplethysmographic chamber (All Medicus, Seoul, Korea) and analyzed. Thebaseline reading was performed for 3 minutes. The enhanced pause(P_(enh)) was calculated according to the manufacturer's protocol [i.e.,(expiratory time/relaxation time−1)χ(peak expiator)/flow/peakinspiratory flow)]. P_(enh) is dimensionless parameter that represents afunction of the proportion of maximal expiratory to maximal inspiratorybox pressure signals and a function of the timing of expiration. Theresults were expressed in percentage increase of P_(enh) according tothe test-infection of methacholine (0, 50 and 100 mg/ml).

Example 13: Immunohistochemistry

In an MPTP-addicted Parkinson's disease animal model, a brine containing0.5% sodium nitrate and heparin (10 U/ml) was transcardially perfusedthrough the experimental and fixed with 4% paraformaldehyde dissolved in0.1M phosphate buffer (PB). The brain was removed and fixed in buffered4% paraformaldehyde overnight, and stored in 30% sucrose solutionmaintained at 4° C. until it became sunk. The brain was fractioned intocoronal slices with a thickness of 30 using a sliding microtome andfrozen. All the fractions in six separated series were collected andsubjected to immunochemical staining. The primary antibodies includethose responding to tyrosine hydroxylase; TH; 1:2000, Pel-freez, BrownDeer, Wis., USA). The stained cells were visualized and then analyzedunder brightfield microscope (Nikon, Tokyo, Japan).

Example 14: Preparation of Recombinant BV-PLA2 and Analysis of itsActivity

In order to prepare polypeptides including a BV-PLA2 amino acid sequencevia recombinant expression, E. coli DH5α and E. coli BL21 (DE3) wererespectively used as the strains for the expression of gene manipulationand protein expression.

14.1. Culturing Bacteria and Purification of Recombinant Proteins

For a large scale production of proteins, 5 ml of a fresh overnightliquid culture of a selected bacteria strain was diluted with 1 L of LBmedium containing ampicillin as an antibiotic. The recombinant E. coliBL21 (DE3) cells containing an expression plasmid were cultured at 37°C. while stirring until the absorbance at 600 nm reached about 0.9. Theexpression of the recombinant PLA2 was induced in a logarithmic growthphase by adding 1 mM isopropyl-β-d-thiogalactoside (IPTG) to the liquidculture. The overnight culture was centrifuged at 4° C. at a rate of3000 rpm for 20 minutes and recovered the cells. The cell pellet wassuspended in a buffer solution A (50 mM sodium phosphate buffer, pH 8.0,and 0.1 mM PMSF and 5 mM β-mercaptoethanol). After pulverizing the cellsusing an ultrasonicator, they were centrifuged at 4° C. at a rate of12000 rpm for 10 minutes, and washed with a buffer solution B (50 mMsodium phosphate buffer, pH 8.0, 2% Triton X-100, 20 mM EDTA, and 0.5 MNaCl) to prepare an insoluble pellet. Then, the resultant was modifiedin a buffer solution C (a buffer solution A containing 8 M urea) for atleast 12 hours. The insoluble cell debris combined with the modifiedrecombinant PLA2 was removed by centrifugation at 4° C. at a rate of12000 rpm for 20 minutes. The histidine-labeled recombinant to PLA2protein was purified via immobilized-metal affinity chromatography usingHis GraviTrap affinity column (GE Healthcare, Piscataway, N.J., USA).The histidine-labeled recombinant PLA2 protein was dialyzed twice at 4°C. using 3 L of 5 mM acetic acid for 12 hours and lyophilized.

14.2. Refolding of Proteins

The lyophilized protein was dissolved in 7 M guanidine-HCl, and thenadded with 0.3 M Na2SO3 (pH 8.3) and a 1/20 volume of Thannhauserreagent for the sulfonification of cysteine thiol. The resultingsolution was dialyzed against 3 L of a mixed solution containing 2 Murea, 4 mM EDTA, 0.1 M NH4Cl, and 20 mM sodium boronate (pH 8.3) forfour hours and then the dialyzed solution was replaced with a freshsolution. After an additional four hours of dialysis, 56 ml of anaqueous solution of 0.5 M cysteine, and 17.5 mL of 0.2 M of cystinesolution dissolved in 1 M HCl solution were added to the dialyzedsolution, and the pH was adjusted to 8.3 using 10 M NaOH. At theappropriate time point, the dialyzed solution was transferred into abeaker wrapped with aluminum foil. When the activity reached the maximumlevel (generally from 18 to 24 hours after addition of cysteine andcystin), the proteins solution was decanted from the agglutinatedprecipitate, and dialyzed at 4° C. with 3 L of SP buffer solution (50 mMTris-HCl, pH 9.0) for 14 hours. The protein purity was confirmed viaSDS-PAGE using 15% (w/v) polyacrylamide for stacking and separatinggels.

14.3. Activity Analysis of Recombinant Proteins

Enzyme activities were determined by an ENZCHECK® phospholipase A2analysis kit (Invitrogen) which uses DOPC as a substrate. According tothe manufacturer's protocol, the enzyme activities of BV-PLA2 andrecombinant PLA2 were analyzed using the ENZCHECK® phospholipase A2analysis kit.

Example 15: Construction of Expression Plasmid and Site-DirectedMutagenesis

The nucleotide sequence of the BV-PLA2 gene used in the presentinvention was inserted in advance into a cloning site of SalI-EcoRI ofpEcoli-Nterm 6×HN vector. The construction of the mutants of the BV-PLA2gene was performed using primer pairs shown below: F-type BV-PLA2(forward): 5′-AAT GTC GAC CAA GTC GTT CTC GGA T-3′ (SEQ ID NO: 12);F-type BV-PLA2 (Reverse): 5′-AAG GAATTC TTATCAATA CTT GCG-3′ (SEQ ID NO:13); A-type BV-PLA2 (Forward): 5′-AAT GTC GAC ATAATATAT CCA GGA-3′(SEQID NO: 14); A-type BV-PLA2 (Reverse): 5′-AAG GAATTC TCA CAG TTT GTAACACTT-3′(SEQ ID NO: 15); B-type BV-PLA2 (Forward): 5′-AAT GTC GAC CAA GTCGTT CTC GGA T-3′(SEQ ID NO: 16); B-type BV-PLA2 (Reverse): 5′-AAG GAATTCTCA CAG TTT GTAACA CTT-3′(SEQ ID NO: 17); C-type BV-PLA2 (Forward):5′-AAT GTC GAC ATAATATAT CCA GGA-3′(SEQ ID NO: 18); C-type BV-PLA2(Reverse): 5′-AAG GAA TTC TTA TCAATA CTT GCG-3′(SEQ ID NO: 19). A singlemutation was introduced into a template, which is a genetic mutant ofBV-PLA2 (H34Q), within the pEcoli-Nterm 6×HN vector using a QuickChangesite-directed mutagenesis kit (Stratagene). The reaction was performedusing the primer pairs shown below: PLA2-H34Q (Forward): 5′-GCATGC TGTCGAACC CAA GAC ATG TGC CCG GAC G-3′(SEQ ID NO: 20); PLA2-H34Q (Reverse):5′-CGT CCG GGC ACA TGT CTT GGG TTC GAC AGC ATG C-3′(SEQ ID NO: 21). Thesubstituted nucleotides were underlined. The validity of the nucleotidesequences of mutants were confirmed by DNA sequencing analysis.

Example 16: Measurement of CD4⁺CD25⁺Foxp3⁺ Regulatory T Cells (Tregs)

Flow cytometry analyses were performed for the samples prepared by themethods described below, and the proportional ratio of CD4⁺CD25⁺Foxp3⁺regulatory T cell (Treg) for each cell was measured accordingly.

16.1. Measurement of CD4+CD25+Foxp3+ Regulatory T Cells (Tregs) fromMurine Splenocytes

Spleens were obtained from six to eight-week old femaleC57BL/6^(Foxp3-EGFP) mice. The spleens were pulverized using a glassslide in the RPMI 1640 medium, and the cells were washed with RPMI 1640medium, and then washed with red blood cell lysis buffer solution(Pharmingen). A 48-well plate with a flat bottom was coated at 4° C.with anti-mouse CD3 antibodies (2.5 mg/ml) overnight. The splenocyteswere seeded into the 48-well plate at a cell density of 4×10⁶ cells/ml,treated with anti-mouse CD28 antibodies (2 mg/ml), and then treated withPBS, PLA2 or recombinant PLA2 for three days and seven days. After thethree day and seven day treatments with the recombinant PLA2, thesingle-cell suspension of the splenocytes were incubated withanti-CD4-APC and anti-CD25-PE monoclonal antibodies using the standardstaining method, and the stained samples were analyzed via a flowcytometer (FACScan Calibur, BD Biosciences). The collection and analysisof data were carried out using a Cell Quest 3.0f software.

16.2. Measurement of CD4⁺CD25⁺Foxp3⁺ Regulatory T Cells (Tregs) fromMurine CD4⁺ T Cells

Murine CD4⁺ Tcells were separated from spleens via MACS CD4 (L3T4)MicroBeads (Miltenyi Biotec Inc., Auburn, Calif., USA). The preparedsingle-cell suspension type cells were combined with the CD4 (L3T4)MicroBeads and incubated at 4° C. for 15 minutes.

After incubation, the cells were washed with MACS buffer and resuspendedin a small volume. The cells were passed through an LS column mounted toVarioMACS magnetic separator. The resultant was washed three to fourtimes and the recovered CD4⁺ cells were resuspended in a complete RPMI1640 medium. The 48-well plate with a flat bottom was coated at 4° C.with anti-mouse CD3 antibodies (2.5 mg/ml) overnight. The CD4⁺ cellswere seeded into the 48-well plate at a cell density of 1×10⁶ cells/ml,treated in the same manner as in Example 16.1, and subjected to flowcytometry analysis.

Example 17: Analysis of Statistical Data

The statistical analysis of data was performed via Prism 5.01 Software(Graph Pad Software Inc., CA, USA). All the values were indicated viamean±S. E. M. (standard error of the mean). The differences between thecontrol group and the groups treated with samples were determinedone-way ANOVA or student t test. In all experiments, p<0.05 wasconsidered to be of statistical significance.

Results of Experiments

Experimental Example 1: In Vitro Direct Increase of CD4⁺CD25⁺Foxp3⁺ Tregby Bee Venom

The CD4⁺CD25⁺Foxp3⁺ Treg population and the expression of Foxp3 in theCD4⁺CD25⁺ Treg in Example 6.1 were analyzed via in vitroimmunofluorescence method. Splenocytes were treated in vitro with beevenom at various concentrations (0, 0.001, 0.01, 0.1, 1, and 10 μg/ml)and the effects according to the concentration were observed (FIGS. 2Aand 2B). The bee venom (1 μg/ml) noticeably increased theCD4⁺CD25⁺Foxp3⁺ Treg (P<0.05) and also increased the expression of Foxp3in CD4⁺CD25⁺ Treg (P<0.001). The bee venom added to the splenocytes canpresumably affect the CD4⁺ T cells directly or through antigenpresenting cells (APC). In order to determine whether bee venom directlyacts on CD4⁺ T cells, the CD4⁺ T cells were separated and treated withbee venom (1 μg/ml) (FIG. 2C). When the CD4⁺ T cells were culturedwithout APC there was an increase in the CD4⁺CD25⁺Foxp3⁺ Treg (P<0.001).Subsequently, it was examined whether bee venom converts the CD4⁺CD25⁻ Tcells into the CD4⁺CD25⁺Foxp3⁺ Tregs. When the CD4⁺CD25⁻ T cells wereadded with bee venom there was no increase in the Treg (FIG. 2D),whereas there was a noticeable increase in the expression of Foxp3 inthe CD4+CD25+ Treg, which was cultured alone after bee venom treatment(FIG. 2E, P<0.001). The above results confirmed that bee venom increasesthe CD4⁺CD25⁺Foxp3⁺ Treg via direct influence on the CD4⁺CD25⁺ Treginstead of Treg induction from the CD4⁺CD25⁻ T cells.

Effect of Treg-Specific Increase of Bee Venom and its In Vivo EfficacyAccording to its Dose

In order to determine the optimum dose of bee venom for in vivoexperiments, the experimental mice were treated daily with variousamounts of bee venom (0, 0.01, 0.1, 1 and 10 mg/kg body weight) for fivedays. Notwithstanding with the various amounts of bee venom, only themice treated with 1 mg/kg of bee venom showed a significant increase ofthe level of CD4⁺CD25⁺ Treg compared to that of the brine-treatedcontrol group (FIG. 3A, P<0.001). The Foxp3 expression in the CD4⁺CD25⁺Treg showed a noticeable increase as is the case with in vitroexperiment (FIG. 3B, P<0.05). Then, the effects of bee venom (1 mg/kg)on other immune cells containing CD4⁺ T cells, CD8⁺ T cells and B cellswere analyzed. While there was no significant increase in the CD4⁺ Tcells, CD8⁺ T cells and B cells as compared to the brine-treated controlgroup, the CD4⁺CD25⁺Foxp3⁺ Treg showed a significant increase (FIGS. 3Cand 3D). The above results confirmed that the dose of bee venom is acrucial factor in its efficacy and that 1 mg/kg may be the optimum dosefor immunological therapy.

Experimental Example 3: In Vitro Myelin-Specific Growth InhibitoryEffect of Bee Venom

The experiment results explained above indicate that bee venom has thepotential therapeutic effect to be used for the treatment of autoimmunediseases. In order to confirm the assumption, firstly, antigen-specificgrowth analysis was performed (FIG. 4). The splenocytes of C57BL/6 miceimmunized with MOG₃₅₋₅₅ were restimulated with MOG₃₅₋₅₅ peptide andcounted the cell number 72 hours thereafter. As a result, it wasconfirmed that the myelin-specific growth of the splenocytes wereinhibited by bee venom in a concentration-dependent manner (P<0.001).

Experimental Example 4: Alleviation of EAE in C57BL/6 Mice by Bee VenomVia Inhibition of Th1/Th17 Differentiation

The possibility of whether the effect of bee venom can be interpretedvia in vivo to experiments was sequentially examined. As the first step,the C57BL/6 mice immunized with MOG₃₅₋₅₅ were treated daily with beevenom (1 mg/kg) from day 0 or day 14 for ten days (FIGS. 5A and 5B). Theresult showed that bee venom lowered severity of diseases as compared tothat of brine-treated mice. This result shows that bee venom has theeffects of prevention and treatment in EAE, which is an establishedanimal model for multiple sclerosis (MS). MS and EAE have been alreadyreported to be mediated by Th1/Th17 reactions. Accordingly, the presentinventors studied the effect of bee venom whether it can inhibitTh1/Th17 differentiation. To this end, they conducted tests on the CD4⁺T cells of EAE-induced mice (FIG. 5C). Both Th1 (P<0.05) and Th17(P<0.05) differentiations were significantly blocked by bee venomtreatment.

Additionally, in order to test whether the Th1/Th17 differentiationaffects the cytokine production, the sera cytokines profiles of the beevenom-treated mice and the brine-treated mice were analyzed on day 24(FIG. 5D). The result showed that the bee venom treatment significantlyreduced the production of other inflammatory cytokines including IFN-γ(P<0.05), IL-17A (P<0.01), TNF (P<0.05) and IL-6 (P<0.05) in the sera.Interestingly, the level of IL-4, which is the main Th2 (IL-4 producingCD4⁺ T cells) cytokine, was also reduced. The above results confirmedthat bee venom has the inhibitory effect against the Th1/Th17differentiation.

Experimental Example 5: Relationship Between the Effect of Bee Venom onEAE Alleviation and CD4⁺CD25⁺ Treg

Experimental Example 1 showed that bee venom increased CD4⁺CD25⁺Foxp3⁺Treg. Additionally, Experimental Example 4 suggested that bee venomalleviates EAE by the inhibition of the Th1/Th17 differentiation. Theabove results imply that the increase in the CD4⁺CD25⁺Foxp3⁺ Treg by beevenom may prevent the Th1/Th17 differentiation thereby improving EAE. Toconfirm the above hypothesis, Treg deficiency was applied with anti-CD25to monoclonal antibodies concurrently with the bee venom treatment onEAE. The previous results showed that the anti-CD25 monoclonal antibodyis suitable for the CD4⁺CD25⁺ Treg deficiency in mice, and since mostCD4⁺CD25⁺ T cells in the present invention exhibit positive result onFoxp3 and thus can be determined by Treg. Additionally, the presentinventors analyzed the progress of EAE and the Th1/Th17 differentiationincluding cytokine production (FIG. 6). The result revealed that therewas no distinct difference in clinical scores, the Th1/Th17differentiation or cytokine production. Conclusively, Treg deficiencyblocks the effect of bee venom on EAE. According to an exemplaryembodiment, the bee venom treatment in mice alleviated EAE via itsdirect effect on the CD4⁺CD25⁺ Treg.

Experimental Example 6: Effect of Bee Venom on Human Treg and Th1/Th17Differentiation

Additionally, the effect of bee venom in humans was examined. In orderto determine whether the level of Treg increases, a fresh PBMC separatedfrom a healthy donor was incubated along with bee venom (1 μg/ml) or PBSfor 24 hours (FIG. 7A). The result revealed that, while the level ofCD4⁺Foxp3⁺ Treg was increased in three out of the four bee venom-treatedsubjects (51.19±17.94% increase vs. PBS), the bee venom treatment in theremaining one subject caused a slight decrease in the level ofCD4⁺Foxp3⁺ Treg (5.16% decrease vs. PBS). Additionally, for thedetection of the Th1/Th17 differentiation, the PBMC was treated with beevenom (1 μg/ml) and anti-CD3/28 antibodies (FIG. 7B). Interestingly, theabove three subjects, which showed an increase in Treg, showed adecrease in Th1 (34.96±4.93% decrease vs. PBS) and Th17 (26.29±8.08%decrease vs. PBS) rates, but the subject, which showed a slight decreasein Treg, did not show a decrease in Th1 (9.71% increase vs. PBS) andTh17 (1.14% increase vs. PBS) rates. The above data confirmed that beevenom can strengthen the inhibitory effect of Treg in humans and alsothat the effect can be applied to each individual subject.

Experimental Example 7: BV-PLA2 as an Active Ingredient of Bee Venom onImmune-Regulation in Mice and Humans

The bee venom of Apis mellifera contains various peptides and proteinsincluding melittin, BV-PLA2, MCD, and apamin. Accordingly, the presentinventors conducted an experiment to examine which component of thevarious bee venom components may exert an effect on Treg in mice. Inparticular, splenocytes, separated from the Foxp3^(EGFP)C57BL/6 mice,were treated with bee venom or each of the components includingmelittin, BV-PLA2, apamin and MCD (FIG. 8). According to the results,bee venom (P<0.05) and BV-PLA2 (P<0.01) induced a noticeable increase inthe CD4⁺CD25⁺Foxp3⁺ Treg, whereas other compounds failed to show thesame. Accordingly, the present inventors confirmed that BV-PLA2 is anactive ingredient of bee venom involved in immune-regulation.Additionally, they also examined the immune-inhibitory effect of BV-PLA2in humans. To this end, freshly separated PBMC was treated with beevenom, BV-PLA2 or PBS and anti-CD3/28 antibodies (FIG. 9). Bee venom andBV-PLA2 showed similar results reducing the populations in Th1 (beevenom: 30.45±10.39%, BV-PLA2: 23.93±6.71% decrease vs. PBS) and Th17(bee venom: 66.04±4.06%, BV-PLA2: 61.80±5.25% decrease vs. PBS).Conclusively, the above results confirmed that BV-PLA2 has animmune-regulation effect both in mice and humans.

Experimental Example 8: In Vitro Effect of BV-PLA2 on InflammatoryCytokines in CD4⁺CD25⁺Foxp3⁺ Treg and Splenocytes

The group treated with BV-PLA2 showed a higher increase inCD4⁺CD25⁺Foxp3⁺ Treg population than the brine-treated group.Additionally, the group treated with BV-PLA2 showed a noticeably higherincrease in the percentage of CD4⁺CD25⁺Foxp3⁺ Treg than thebrine-treated group (FIG. 10). The levels of IL-2, the Th1 cytokinecapable of activating B cell by Th2, were more significantly decreasedin the culture supernatant of the group treated with BV-PLA2 than in thebrine-treated group. The above decrease in IL-2 indicates that BV-PLA2can inhibit the inflammation caused by B cell immune responses. Thelevels of IL-10, a kind of Treg cytokines, were substantially increasedin the group treated with BV-PLA2 than in the brine-treated group.However, BV-PLA2 failed to show any effect of statistical significance(FIG. 11).

Experimental Example 9: Effect of BV-PLA2 on CD4+CD25+Foxp3+ Treg inOVA-Induced Asthma Group

In alveolar cells separated from an OVA-induced asthma mouse model, theOVA group showed a higher decrease in the CD4⁺CD25⁺Foxp3⁺ Tregpopulation than the CON group. The (OVA+PLA2) group showed a higherincrease in CD4⁺CD25⁺Foxp3⁺ Treg population than the OVA group. Theabove results confirmed that BV-PLA2 can improve reduced Treg inallergic asthma (FIG. 12).

Experimental Example 10: Effect of BV-PLA2 on Th2 Cytokines (IL-4 andIL-13) in BALF

The cytokine levels in the BALF of the control group and each mousemodel of the experimental groups were measured. According to the result,the OVA, OVA-T and (OVA-T+PLA2) groups showed a higher increase in thelevel of Th2 inflammatory cytokine than the (OVA+PLA2) group and thecontrol group (FIG. 13). BV-PLA2 could reduce the production of theinflammatory cytokine increased due to OVA treatment, but in theTreg-deficient group, BV-PLA2 treatment failed to show the aboveinhibitory effect. Accordingly, it was confirmed that Treg is involvedin the inhibition of inflammatory cytokine secretion by BV-PLA2.

Experimental Example 11: Effect of BV-PLA2 on IgE Titer in Sera

The OVA group showed a noticeably higher increase in IgE concentrationin the serum than the CON group. This indicates that the asthmainduction in an animal model of the present invention conducted inExample 1.2 was successfully performed. The (OVA-T+PLA2) group showed asignificantly higher decrease in the serum IgE concentration than in theOVA, OVA-T and (OVA-T+PLA2) groups. BV-PLA2 could inhibit the increasein serum IgE concentration in OVA-induced allergic asthma mice, but theeffect of BV-PLA2 failed to show in the Treg-deficient group (FIG. 14).This suggests that the inhibitory effect of BV-PLA2 against the increaseof serum IgE concentration may occur via Treg activation.

Experimental Example 12: Effect of BV-PLA2 on Total Cells andInflammatory Cells in BAL

The OVA group showed a significantly higher increase in the number oftotal cells, eosinophils, and lymphocytes than the CON group. Thisindicates that the asthma induction in an animal model of the presentinvention conducted in Example 1.2 was successfully performed. The(OVA+PLA2) group showed a significantly higher decrease in the number oftotal cells, eosinophils, and lymphocytes than in the OVA, OVA-T and(OVA-T+PLA2) groups. BV-PLA2 could inhibit the increase of inflammatorycells such as the total BAL cells, eosinophils, lymphocytes inOVA-induced allergic asthma mice, but the effect of BV-PLA2 failed toshow in the Treg-deficient group (FIG. 15). This suggests that theinhibitory effect of BV-PLA2 against the increase of the total BALcells, eosinophils, lymphocytes may occur via Treg activation.

Experimental Example 13: Effect of BV-PLA2 on the Change in Lung Shapein an OVA-Induced Asthma Model

In order to analyze the effect of PLA2 on the histologicalcharacteristics of asthma, the lung tissues of OVA-induced allergicasthma mice were subjected to H&E and PAS staining. According to theresult, the lung tissue slices obtained from the mice exposed to OVAshowed an airway inflammation, and pulmonary infiltration witheosinophils around the bronchial was observed (FIG. 16). In contrast,the lung tissue slices obtained from the mice treated with BV-PLA2showed a decrease in airway inflammation. The periodic Acid Schiff(PAS)-positive mucus-count goblet cells present around the bronchialairway were detected in the OVA, OVA-T and (OVA-T+PLA2) groups.Meanwhile, BV-PLA2 treatment significantly reduced PAS-positive gobletcells around the bronchial airway (FIG. 17). The discovery suggests thatBV-PLA2 has the potential of reforming an effective airway.

Experimental Example 14: Effect of BV-PLA2 on Airway Hyperresponsivenessin an OVA-Induced Asthma Model

In order to confirm the inhibitory effect of BV-PLA2 against airwayhyperresponsiveness, P_(enh) values were measured. At concentrations of50 mg/ml and 100 mg/ml of methacholine, the OVA, OVA-T and (OVA-T+PLA2)groups showed a noticeably higher increase in P_(enh) value than in theBV-PLA2 group. Meanwhile, the group treated with BV-PLA2 showed adecrease in the increased P_(enh) value at 50 mg/ml and 100 mg/mlconcentrations of methacholine relative to the CON group of theOVA-induced asthma mice (*P<0.05, **P<0.01; FIG. 18).

Experimental Example 15: Effect of BV-PLA2 on Protection of Dopaminergic(DA) Neurons in Substantia Nigra (SN) in a Parkinson's Disease AnimalModel

Seven days after MPTP-addiction, the brain slices of an MPTP-inducedParkinson's disease animal model prepared in Example 1.3 were acquired,and DA neurons were immunostained with anti-TH antibodies. The BV-PLA2treatment noticeably increased the number of TH⁺ neurons in SN to 32%compared to that of the mice in the MPTP-addicted control group (P<0.01;FIG. 1).

BV-PLA2 treatment induced the activation of microglia after theinfiltration of CD4⁺ T cells in SN. According to a recent report,CD4⁺CD25⁺ Treg mediates the protection of neurons by inhibitingmicroglia reaction in an MPTP-induced PD model. In order to confirmwhether Treg is involved in the neuron protection by BV-PLA2,MPTP-addicted mice were intraperitoneally injected with anti-CD25antibodies (1 mg/kg) one day prior to MPTP to administration, andthereby reduced the number of Treg cells. The BV-PLA2 treatment on themice with a reduced number of Treg cells failed to reduce the DAneuronal death unlike the MPTP-addicted mice.

Experimental Example 16: Gene Synthesis and Construction

The BV-PLA2 used in the present invention accounts for about from 10% to20% of bee venom components, and the recombinant BV-PLA2 was prepared byRNA extraction from the honey bee (Apis mellifera), followed by cDNAsynthesis, and PCR amplification using BV-PLA2-specific primers. Theamplified gene was replicated into SalI and EcoRI restriction sites inthe pEcoli-Nterm 6×HN vector. The recombinant plasmid was transformedinto E. coli strain DH5α and the recombinant plasmid DNA was extractedtherefrom, and confirmed whether it was the correct recombinant BV-PLA2via sequencing analysis of the recombinant plasmid DNA. For alarge-scale production of the recombinant BV-PLA2, the recombinantplasmid were introduced into E. coli strain BL21 via transformation. Thefour types (F, A, B, and C) of BV-PLA2 used in the experiments werereplicated into the pEcoli-Nterm 6×HN vector in the same manner asdescribed above (FIG. 20).

Experimental Example 17: Purification of Recombinant BV-PLA2 and itsEnzyme Activity

The various types of the recombinant BV-PLA2 prepared in the presentinvention were extracted in the same manner as described above, andconfirmed via SDS-PAGE using 15% (w/v) polyacrylamide. As a result, itwas confirmed that native secretory type BV-PLA2 is a protein with asize of 134 AA, F-type recombinant BV-PLA2 with 201 AA, A-typerecombinant BV-PLA2 with 133 AA, B-type recombinant BV-PLA2 with 166 AA,and C-type recombinant BV-PLA2 168 AA (FIG. 21A; A-type not shown).

Enzyme activities were analyzed for the above three types of recombinantBV-PLA2s and the native BV-PLA2 using an ENZCHECK® phospholipase A2analysis kit (Invitrogen), which utilizes DOPC substrate, as describedin Example 14.3. According to the measurement, the activity of thenative BV-PLA2 was 8 U/MW, and that of the F-type recombinant BV-PLA2was 2 U/MW, but the enzyme activity of the B-type recombinant BV-PLA2was not measured. In contrast, the enzyme activity in the C-typerecombinant BV-PLA2 measured was shown to be similar to that of thenative BV-PLA2, which is a positive control group (FIG. 21B).

Experimental Example 18: Effect of Recombinant BV-PLA2 onCD4⁺CD25⁺Foxp3⁺ Regulatory T Cell (Treg)

In order to examine whether the recombinant BV-PLA2 can control theCD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) in splenocytes, spleens wereobtained from six to eight-week old C57BL/6^(Foxp3-EGFP) mice. Thesplenocytes were stimulated with anti-mouse CD3 antibodies andanti-mouse CD28 antibodies, and treated with the native BV-PLA2 and therespective recombinant BV-PLA2 at a concentration of 1 mg/ml for threedays, stained with anti-CD4-APC and anti-CD25-PE monoclonal antibodies,and were measured using a flow cytometer. The native BV-PLA2 showed atwo-fold or higher of increase than that of the control group (1.66%),whereas F and B types showed no effect compared to that of the controlgroup. In contrast, the C-type recombinant BV-PLA2 was shown to have3.96% thus confirming that it is effective on the increase ofCD4⁺CD25⁺Foxp3⁺ regulatory Tcells in the splenocytes (FIGS. 22A through22E).

Additionally, in order to examine whether the recombinant BV-PLA2controls the CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) in CD4⁺ T cells,spleens were obtained from six to eight-week old C57BL/6^(Foxp3-EGFP)mice. Only the CD4⁺ T cells were separated from the splenocytes via MACSCD4 (L3T4) MicroBeads, stimulated with anti-mouse CD3 antibodies andanti-mouse CD28 antibodies, treated with the native BV-PLA2 and therespective recombinant BV-PLA2 at a concentration of 1 mg/ml for threedays, stained with anti-CD4-APC and anti-CD25-PE monoclonal antibodies,and were measured using a flow cytometer. The native BV-PLA2 showed atwo-fold or higher of increase than that of the control group (2%),whereas F and B types showed no effect compared to that of the controlgroup. In contrast, the C-type recombinant BV-PLA2 was shown to have 4%thus confirming that it is effective on the increase of CD4⁺CD25⁺Foxp3⁺regulatory Tcells in the splenocytes (FIG. 23).

Experimental Example 19: Preparation of C-Type Mutant (H34Q) RecombinantBV-PLA2 and its Enzyme Activity

The enzyme active site of the C-type mutant (H34Q) recombinant BV-PLA2(SEQ ID NO: 11), prepared by the method described in Example 15 of thepresent invention, was modified from histidine to glutamine, and theC-type mutant (H34Q) recombinant BV-PLA2 was extracted in the samemanner as described above, and confirmed via SDS-PAGE using 15% (w/v)polyacrylamide. The protein size of the C-type recombinant BV-PLA2 andthe C-type mutant (H34Q) recombinant BV-PLA2, i.e., 171 AA, wasconfirmed via SDS-PAGE (FIG. 24A).

The enzyme activity of the C-type mutant (H34Q) recombinant BV-PLA2prepared above was measured using ENZCHECK® phospholipase A2 analysiskit (Invitrogen), which utilizes DOPC as a substrate. According to themeasurement, the activity of the native BV-PLA2 was 8 U/MW, and theactivity of the C-type mutant (H34Q) recombinant BV-PLA2 was shownsimilar to that of the native BV-PLA2. However, the measured activity ofthe C-type mutant (H34Q) recombinant BV-PLA2 was low (FIG. 24B).

Experimental Example 20: Effect of C-Type Mutant (H34Q) RecombinantBV-PLA2 on CD4+CD25+Foxp3+ Regulatory T Cells (Treg)

In order to examine whether the C-type mutant (H34Q) recombinant BV-PLA2prepared above can control the CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg)in the splenocytes and the CD4⁺ T cells, spleens were obtained from sixto eight-week old C57BL/6^(Foxp3-EGFP) mice. The sample of thesplenocytes and the sample obtained by separating only CD4⁺ T cells fromthe splenocytes via MACS CD4 (L3T4) MicroBeads, were respectivelystimulated with anti-mouse CD3 antibodies and anti-mouse CD28antibodies, treated with the native BV-PLA2 at a concentration of 1mg/ml, and the C-type recombinant BV-PLA2 and the C-type mutant (H34Q)recombinant BV-PLA2 at a concentration of 2 mg/ml, respectively, forseven days, stained with anti-CD4-APC and anti-CD25-PE monoclonalantibodies, and were measured using a flow cytometer. In thesplenocytes, the native BV-PLA2 showed an increase by 28%, and theC-type recombinant BV-PLA2 showed an increase by 45%, respectively,compared to that of the control group, whereas the C-type mutant (H34Q)recombinant BV-PLA2 showed no effect compared to that of the controlgroup (FIG. 25A). Meanwhile, in the CD4⁺ T cells, the native BV-PLA2showed an increase by 7%, the C-type recombinant BV-PLA2 showed anincrease by 9%, respectively, compared to that of the control group,whereas the C-type mutant (H34Q) recombinant BV-PLA2 showed no effect onthe increase of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg), compared tothat of the control group (FIG. 25B).

The invention claimed is:
 1. A method for treating rheumatoid arthritis,multiple sclerosis or psoriasis comprising: administering an effectiveamount of a composition comprising an isolated polypeptide to a subjecthaving rheumatoid arthritis, multiple sclerosis or psoriasis, whereinthe polypeptide comprises the amino acid sequence of SEQ ID NO:6.